MicroRNAs (miRNAs) are small non-coding RNAs (typically about 22 nucleotides in length) that were discovered as important post-transcriptional regulators of gene expression in metazoans (Bartel, 2004). While expression of miRNAs are critical in various physiological processes (Harfe, 2005; Miska, 2005; Carthew, 2006; Lindsay, 2008), dysregulation of miRNAs are implicated in pathologies of many human diseases such as cancer (Visone & Croce, 2009), muscle disorders (Chen et al., 2009a) and neurodegeneration (Hebert & De Strooper, 2009).
Recently, mature miRNAs were found to be remarkably stable in blood, and thus hold great promise as potential noninvasive biomarkers of human diseases (Chen et al., 2008; Mitchell et al., 2008). To date, hundreds of unique miRNAs have been identified in many species and each of these is predicted to regulate diverse target genes (Bartel, 2009). With the continual discovery of more miRNAs by both in silico prediction and in vivo validation (Mendes et al., 2009), profiling of miRNA expression remains an essential tool not only for assessment of distribution and regulation of miRNAs but also for identification of novel biomarkers and potential therapeutic targets.
Mature miRNAs can be detected by either direct or indirect methods. Although direct detection methods (eg. fluorescent, colorimetric and electrical-based methods) can minimize variations introduced during sample measurements, these methods are limited by low assay sensitivity and poor discrimination among miRNA homologs (reviewed in Hunt et al., 2009).
Indirect detection methods include primarily Northern blotting, microarray and reverse transcription PCR (RT-PCR). Although widely used, both Northern blotting and microarray are semi-quantitative and suffer from poor sensitivity and require large amounts of starting RNA. Although microarrays offer high-throughput detection of miRNAs and the potential capability of absolute quantification (Bissels et al., 2009), a recent study showed that real-time PCR remains superior in sensitivity and specificity in comparison (Chen et al., 2009b). Recent attempts to measure miRNA with isothermal methods have met with some success but are labor intensive (Cheng et al., 2009; Yao et al., 2009).
To date, real-time RT-PCR remains the most sensitive and efficient method for quantification of RNA species. TaqMan probe-based real-time RT-PCR has been reported and widely used for efficient and specific detection of miRNAs. However, due to an additional probe hydrolysis step, TaqMan assays were not compatible with fast thermo-cycling protocols for rapid detection of miRNAs. Furthermore, with the escalating identification of hundreds of candidate miRNAs by deep sequencing (Bar et al., 2008; Goff et al., 2009), design of TaqMan probe for each of the novel miRNA is not only cost-prohibitive but also technically challenging and faces practical difficulties (Varkonyi-Gasic et al., 2007).
Attempts have been made to improve miRNA detection without reliance on fluorescent probes (Raymond et al., 2005; Shi & Chiang, 2005; Sharbati-Tehrani et al., 2008), however, these assays usually involved multiple sample processing steps (Shi & Chiang, 2005) and suffered from limited dynamic range of detection (Raymond et al., 2005) and/or poor specificity against homologous miRNAs (Raymond et al., 2005; Shi & Chiang, 2005; Sharbati-Tehrani et al., 2008). A common strategy of these assays and some other TaqMan assays (Varkonyi-Gasic et al., 2007; Yang et al., 2009) is to use universal or common reverse PCR primer and/or fluorescent probe for amplification and detection of multiple miRNAs. In such assays, specificity of real-time PCR is only achieved by the forward PCR primer, which is not sufficient for discrimination of many homologous miRNAs. Furthermore, it is yet to be determined whether these assays are capable of rapid, multiplexed and direct detection of miRNAs without RNA isolation.
While the number of microRNAs in various genomes are still being identified, the number of different miRNA is expected to be as many as a few thousand. Since the Nobel prize was awarded in 2006 for RNA interference (A. Z. Fire & Craig C. Mello), the demand for assays to detect miRNA has steadily increased.
Thus, there exists a need for alternative methods of detecting a target RNA in a sample, including an miRNA.